Integrated multi-omics unveil the impact of H-phosphinic analogs of glutamate and α-ketoglutarate on Escherichia coli metabolism

Desmethylphosphinothricin (L-Glu-γ-P_H) is the H-phosphinic analog of glutamate with carbon-phosphorus-hydrogen (C-P-H) bonds. In L-Glu-γ-P_H the phosphinic group acts as a bioisostere of the glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5'-phosphate-dependent α-decarboxylase. In addition, the L-Glu-γ-P_H decarboxylation product, GABA-P_H, is further metabolized by bacterial GABA-transaminase, another pyridoxal 5'-phosphate-dependent enzyme, and succinic semialdehyde dehydrogenase, a NADP⁺-dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, is succinate-P_H, the H-phosphinic analog of succinate, a tricarboxylic acid cycle intermediate. Notably, L-Glu-γ-P_H displays antibacterial activity in the same concentration range of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-P_H was shown to display an even higher efficacy, likely as a consequence of an improved penetration into the bacteria. Herein, to further understand the intracellular effects of L-Glu-γ-P_H, ¹H NMR-based metabolomics, and LC-MS-based shotgun proteomics were used. This study included also the keto-derivative of L-Glu-γ-P_H, α-ketoglutarate-γ-P_H (α-KG-γ-P_H), which also exhibits antimicrobial activity. L-Glu-γ-P_H and α-KG-γ-P_H are found to similarly impact bacterial metabolism, although the overall effect of α-KG-γ-P_H is more pervasive. Notably, α-KG-γ-P_H is converted intracellularly into L-Glu-γ-P_H, but the opposite was not found. In general, both molecules impact the pathways where aspartate, glutamate, and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response, and deprive cells of nitrogen. This work highlights the multi-target drug potential of L-Glu-γ-P_H and α-KG-γ-P_H and paves the way for their exploitation as antimicrobials.